Process for the preparation of derivatives of 6-amino-penicillanic acid



United States Patent Ofi 3,094,519 Patented June 18, 1963 ice 3,694,519 PROCESS FOR TEE PREPARATEQN F BERN TIVES 0F -AMlNfi-PENECIILLANIC ACilD Joyce R. Luttinger, Syracuse, Joseph Lein, Fayetteviile,

and Alexander Gourevitch, Syracuse, N.Y., assignors to Bristol-Myers Company, New York, N.Y., a corporation of Delaware No Drawing. Filed Jan. 16, 1961, Ser. No. 82,694

4 Claims. (Ci. 260-239.1)

This invention relates to a process for the preparation of u-aminobenzylpenicillin and, more particularly, to a chemical process involving the reaction of 6-aminopenicillanic acid and a thioeste-r of Z-phenylglycine.

a-Aminobenzylpenicillin is a compound which is known in the technical literature, having been described in South African Patent No. 59/3827, dated September 29, 1959. The preparation of a-a minobenzylpenicillin as described in that patent (see particularly Example 11) involves the preparation of a-arninophenylacetic acid, the reaction of that acid so as to protect the amino group, e.g., as with benzoxycarbonyl chloride to form ot-carbobenzoxyarninophenylacetic acid, the formation of a mixed anhydride of that compound, the reaction of the mixed anhydride with 6-aminopenicillanic acid, the subsequent removal of the protecting group from the amino group by hydrogenolysis and finally the purification of the product. Such a method has been found to be very difficult to carry out on a commercial scale and, moreover, has the disadvantage of being very costly, particularly in the hydrogenolysis step which requires substantial amounts of hydrogenation catalyst.

The invention is therefore broadly directed to an improved method for the preparation of a-amincbenzylpenicillin and a-alkylaminobenzylpenicillins. It is a general object of the invention to provide a process for the preparation of warninobenzylpenicillin comprising the reaction of 6-aminopenicillanic acid and thioesters of 2-phenylglycine. It is a particular object of this invention to provide a commercially useful method by which 6-aminopenicillanic acid may be reacted with thioesters of 2- phenylglycine to produce ot-aminobenzylpenicillin and either of the diastereoiscmers thereof in high yields substantially free of contaminants with a minimum of manipulative operations. Other objects and advantages of the invention will become apparent in the following detailed description and illustration of the invention.

According to the present invention, G-aminopenicillanic acid is brought into contact in a suitable liquid reaction medium with a member selected from the group consisting of compounds having the formulae and i c zuos-onnnooort wherein R and R are each members selected from the group consisting of hydrogen, hydroxy, lower alkyl, lower alkoxy, chioro, bromo, iodo, fluoro, nitro, sulfamyl, cyclopentyl, cyclohexyl, acylamino, lower alkylamino, di lower alkylamino, lower alkylthio, benzyl and trifiuorornethyl, wherein R is a lower alkyl radical, and wherein n is an integer from 1 to 6 inclusive; and the acid addition salts thereof, including such mineral acid addition salts as the hydrochloride, hydroiodide, hydrobromide, sulfate, sulfamate and phosphate and the organic acid addition salts such as the maleate, acetate, citrate, mal-ate, oxalate, succinate, benzoate, tartrate, fumarate, mandelate, ascorbate and the like. The term lower alkyl as used herein means both straight and branched chain saturated aliphatic hydrocarbon radicals having from one to six carbon atoms, such as, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, t-butyl, etc. The term C H as used herein means a lower alltylene radical, i.e., saturated bivalent straight and branched chain aliphatic hydrocarbon radicals having not more than six carbon atoms such as methylene, ethylene, propylene, l-methylethylene, 2-methylpropylene, etc.

A preferred process for the present invention comprises contacting 6-aminopenicillanic acid and a member selected from the group consisting of S-(2-phenylglycyl)- thiophenol hydrochloride and s-(a-aminophenylacetyl)- thioacetic acid hydrochloride in an aqueous acidic reaction medium and recovering tt-aminobenzylpenicillin from the reaction mixture. As noted below, the thioester of 2- pheny-lglycine can be either of the pure diastereoisomers or a mixture or the diastereoisorners of the thioester.

The preferred aqueous acidic reaction medium is an aqueous solution of acetic acid containing from about 25% to 60% of acetic acid. When lower concentrations (than 25%) of acetic acid are used, it is desirable to acidity (e.g., with hydrochloric acid) the solution to a pH of from about 1.5 to about 3, preferably to about pH 2. Use of an aqueous buffer solution having a pH of from about 5.5 to 6.0 results in the yields that are somewhat lower than those obtained with the preferred reaction medium. The reaction time in the preferred reaction medium can be varied widely from only a few minutes (3 to 15 minutes) to several hours, e.g., three or four hours. The temperature of the reaction can also be varied widely from temperatures several degrees below 0 C. to about 50 C. The preferred reaction temperature is about 0 C. When the reaction is conducted in an aqueous buffer solution at a pH of from about 5.5 to 6.0, the preferred reaction time is from about one to four hours. The use of a citrate buifer to control pH is preferred though other buffers which adequately control the pH to the desired range may also be used, e.g., phosphate, succinate and acetate buffers, etc. Other conventional means of maintaining the desired pH of the reaction mixture can also be employed, e.g., the periodic or continuous addition of acids or bases. Agitation of the reaction mixture is desirable in order to promote contact of the 6-aminopenicillanic acid and the thioesters, some of which are sparingly soluble in the reaction media. Recovery of the penicillin from the reaction media can be accomplished by conventional means, e.g., according to the procedures set forth in South African Patent No. 59/3827.

The thioesters of Z-phenylglycine and 2-alkylamino- Z-phenylacetic acid which are reacted with 6-aminopenicillanic acid in the process of this invention are readily prepared according to methods which are described in the technical literature and which are illustrated in Examples 1 and 2 below. Z-phenylglycine and many of the thiophenols and mercaptoalkanoic acids are commercially available. Z-phenylglycine and 2-allrylamino-2-phenylacetic acid can be prepared by the Strecker reaction which is described in Merck Index, 7th edition, page 1471, and in articles mentioned therein. Specific techniques for the preparation of some thioesters of certain amino acids are described in an article by T. Wieland et al. in Ann. 588, page 15 et sub. (1954).

The a-carbon atom of the 2-phenylglycyl radical is an asymmetric carbon atom and the thioesters of 2-phenylglycine can therefore exist in two optically active isomeric forms, [the D-() and L-(+) diastereoisomers], as well as the optically inactive DL form which is a mixture of the two optically active forms; either of such isomeric forms of the thioesters can be utilized in the process of this invention to produce either of the two isomers of ot-aminohenzylpenicillin and the mixture of the isomers of the thioesters can be used to produce DL-ot-aminobenzylpenicillin.

The process of the present invention can be employed to prepare the two diastereoisomers of ot-aminobenzylpenicillin by first preparing an optically pure isomer of 2-phenylglycine, e.g., separating the optically active forms of DL-2- phenylglycine by way of their salts with optically active bases such as quinine, brucine, etc., then preparing a thioester of either such optically active form of 2-phenylglycine and then employing the appropriate form in the acylation of 6-aminopenicillanic acid. Thus DL-Z-phenylglycine may be resolved to produce D-Z-phenylglycine and L-2-phenylglycine, each of which acids can be used to form an appropriate thioester, and then each of the thioesters may be reacted with 6-aminopenicillanic acid to produce D-( )-a-aminobenzylpenicillin and L-(+)-ot-aminobenzylpenicillin, respectively.

A mixture of the isomers of 2-phenylglycine can be resolved according to methods which are described in detail in the technical literature, particularly in the following references: Betti and Mayer, Ber., 41, 2073 (1908); Ingersoll and Adams, I. Am. Chem. Soc., 47, 1168 (1925); Reihlen and Knopfle, Ann., 523, 199 (1936); Reihlen, Knopfle and Snopper, Ann., 534, 247 (1938); and Kuna, Ova-kirinan and Levene, J. Biol. Chem., 137, 334 (1941).

Various embodiments of the invention are illustrated in the following examples. In the following examples, particularly in the tables, 6-aminopenicillanic acid is referred to as 6-APA, DL-a-aminobenzylpenicillin is referred to as ABP and S-(DL-Z-phenylglycyl)-thiophenol hydrochloride is referred to as Thiophenol and S-(DL-OC- aminophenylacetyl)-thioacetic acid hydrochloride is referred to as thioacetic acid.

EXAMPLE 1 Preparation of S- DL-Z-Phenylglycyl) -Thiophenol Hydrochloride A suspension of DL-Z-phenylglycine (40.0 gms.; 0.264 mole) in one liter of dichloromethane and 4 ml. of dimethylformamide is prepared and cooled to 5 C. and phosphorus pentachloride (80.0 gms.; 0.384 mole) is added to the suspension which is then stirred for fifteen minutes in an ice-bath and thereafter for one hour at room temperature. The product precipitates, is collected by filtration, washed twice with dichloromethane and thereafter washed once with dry ether, dried in vacuo over phosphorous pentoxide, and found to weigh 30 gms. The product is added to the thiophenol (44.1 gms.; 0.4 mole) and 100 ml. of dimethylformamide while the mixture is cooled in ice. The reaction mixture is cooled and shaken for ten minutes until solution is complete and thereafter maintained at room temperature for 1% hours. The reaction mixture is then diluted to about 1.5 liters with ether, decanted and the remaining gummy product is washed with dry ether whereupon crystallization begins. The ether is decanted and the product covered with 200 ml. acetone whereupon crystallization of the product is completed. The crystalline product, S-(DL-Z-phenylglycyl)-thiophenol hydrochloride, is collected by filtration, washed with acetone and ether, dried in vacuo over P 0 and found to weigh 19.2 gms., to have a melting point of 212214 C. (with decomposition) and to have the following elemental analysis: Calculated for C H ClNOS: C, 60.01%; .H, 5.05%. Found: C, 58.71%; H, 5.73%.

EXAMPLE 2 Preparation of S-(DL-a-Aminophenylacetyl)-Thi0acetic Acid Hydrochloride A suspension of DL-2-phenylglycine (10 gms; 0.066 mole) in 250 ml. dichloromethane is formed and there is added to the suspension 1 ml. dimethylformamide and phosphorus pentachloride (20 gms.; 0.096 mole) with cooling in ice. The suspension is stirred for 20 minutes at ice bath temperature and thereafter for 45 minutes at room temperature. The resulting white solid precipitate is filtered quickly and washed twice with dichloromethane and then with dry ether and is dried in vacuo for two hours over P 0 The product which is found to weigh 6 gms. (0.029 mole) is added to 5.5 gms. (0.06 mole) of mercaptoacetic acid and 30 ml. of dry dimethylformamide is added to the reaction mixture with cooling. The mixture is maintained for one hour at room temperature and thereafter diluted to the cloud point with dry ether and stored for three days at 5 C. The white crystalline product, S-(DL-ot-aminophenylacetyl)-thioacetic acid hydrochloride, is then collected by filtration, dried in vacuo over P 0 and is found to weigh 0.5 gm., to melt at 185 C., to decompose at 210 C., and to have the following elemental analysis: Calculated for C H ClNO s: C, 46.0%; H, 4.65%; S, 1.23%. Found: C, 48.72%; H, 6.72%; S. 1.26%.

EXAMPLE 3 Preparation of ot-Aminobenzylpenicillin A reaction mixture consisting of S-(DL-2-pheny1glycyD-thiophenol hydrochloride (25 mg.) and 6-aminopenicillanic acid (25 mg.) in 2.5 ml. of water and 2.5 ml. of a 0.2 M sodium succinate-succinic acid butter solution having a pH of 5.5 (the reaction mixture having a pH of 5.3) is vigorously agitated at 37 C. for six hours. After three hours a one m1. sample of the reaction mixture which had then attained a pH of 5.0 is withdrawn and after six hours another one ml. sample of the reaction mixture which had then attained a pH of 4.8 is withdrawn. Each sample is diluted immediately after its withdrawal with a dipotassium phosphate buffer solution (0.1 M; pH 6.2) to produce samples of SOO-fold and 3,000-fold dilution for bioassay and the samples at each dilution are immediately cooled to minimize further reaction. Each sample is then assayed against Staph. aureus to determine the concentration therein of ot-aminopenicillanic acid. It is determined from the assay that each three hour sample contains about 625 meg/ml. of ot-aminobenzylpenicillin and each six hour sample contains 675 meg/ml. of a-aminobenzylpenicillin.

The foregoing procedure was repeated with a reaction mixture containing the same quantities of the thiophenol ester and 6-aminopenicillanic acid in 0.5 ml. of the same buffer, together with 2.5 ml. of water and 2 ml. of Water saturated with urea, the urea being used to aid in the solubilization of the ester. It was determined by sampling and assay in the manner described above that after three hours the reaction mixture which had a pH of 4.5 contained 525 meg/ml. of oc-aminobenzylpenicillin; after six hours the pH of the reaction mixture had dropped to 4.1 and no measurable amount of penicillin was found therein.

EXAMPLE 4 Preparation of a-Aminobenzylpenicillin A reaction mixture consisting of S-(DL-a-arninophenylacetyl)-thioacetic acid hydrochloride (10 mg.) and 6- aminopenicillanic acid (10 mg.) in 1.0 ml. of water and 1.0 ml. of a 0.4 M sodium citrate-citric acid bufier solution having 21 111-1 of 5.5 (the reaction mixture initially having a pH of 6.05) is vigorously agitated at 37 C. for 6% hours. After 3% hours a one ml. sample of the reaction mixture which had then attained a pH of 5.6 is withdrawn and at 6% hours another one ml. sample is withdrawn and found to have a pH of 5.7. Each sample is diluted immediately after its withdrawal with a dipotassium phosphate buffer solution (0.1 M; pH 6.2) to produce samples of 500 fold and 3,000-fold dilution for bioassay and the samples at each dilution are immediately cooled to minimize further reaction. Each sample is then assayed against Staph. aureus to determine the concentration therein of u-aminopenicillanic acid. The assay results (uncorrected) show that the 3% hour sample contains about 650 meg/ml. of a-aminobenzylpenicillin and the 6 /4 hour sample contains 700 meg/ml. of DL-a-aminobenzylpenicillin. The corrected values for concentration of the penicillin are about one-half of the uncorrected values.

EXAMPLE 5 Preparation of a-Aminobenzylpenicillin Using Various pHs, Bufiers and Concentrations of Reagents A number of experiments (Expt. 1-22) are conducted using reaction mixtures having the composition set forth in Table I below:

TABLE I Expt. 6-APA, Tliiophe- Bnfier solution mg. n01, mg.

Ml. pH

10 10 2 6. 0.2 M citrate. 10 10 2 5.0 0. 2 M succinate. 10 10 2 5. Do. 10 2 4.0 0.2 M acetate. 10 10 2 4. 5 o. 10 10 2 6. 0 0.2 M phosphate 10 10 2 7. 0 Do. 10 10 2 8.0 Do. 10 2 5. 5 0.2 M citrate. 10 2 5. 5 D0. 20 10 2 5. 5 D0. 14 10 2 5. 5 Do. 20 20 2 5. 5 Do. 40 40 2 5. 5 Do. 10 0 2 5. 5 D0. 20 0 2 5. 5 Do. 40 0 2 5. 5 Do.

0 40 2 5. 5 Do. 10 10 2 5. 5 D0. 10 1O 2 5. 5 Do.

duce samples of 10-fold and 100-fold dilution for bioassay and the samples at each dilution are immediately cooled to minimize further reaction. The concentration of a-aminobenzylpenicillin in the sample is determined by bioassay against B. subtilis by the disc method. The results are set forth in Table II.

TABLE II pH at- Concentration of .ABP,

meg. ml. Sample from expt.

0 hours 4% hours 4% hours 24 hours 5. 75 5. 1, 000 880 5. 05 4. 650 780 5. 3 5. 4 800 850 3. 95 3. 9 02500 280 4. 4 4. 42 Ca700 500 5. 65 5. 42 (321800 730 6. 6 6. 65 Ca470 300 6. 95 7. 2 Ca430 175 5. 5 5. 58 1, 000 1, 400 5.45 5. 5 900 1, 300 5. 53 5. 52 1, 650 1, 400 5. 32 5. 25 1, 850 l, 600 5. 43 5. 45 1, 550 1,800 5. 3 5. 2 3, 2, 900 5. 58 5. 7 420 500 5. 48 5. 55 310 5. 4 5. 35 011650 5. 72 5. 65 N NR 5. 68 5. 6 NR NR 5. 5 5. 45 NR NR 5. 45 5. 7 1, 000 1,500 5. 55 5. 7 900 2, 200

NOTE.NR as used above means No Ring and indicates no measurable amount of a-aminobenzylpemcillm.

From the foregoing data, it is apparent that in this series of experiments the yield of DL-a-aminobenzylpenicillin is greatest where a sodium citrate-citric acid buffer is used and the pH of the reaction is maintained at about 5.5 to 6.0. The data also indicates that yields are not materially increased by increasing the proportionate amount of the thiophenol ester or 6-aminopenicillanic acid or by increasing the reaction time to 24 hours.

EXAMPLE 6 Efiect of Reaction Conditions and Reagent Concentrations on Reaction of o-Aminopenicillanic Acid and S-(DL-Z-Phenylglycyl)-Thi0phen0l Hydrochloride The effect of variations in the time, temperature, pH, the buffer system employed and concentration of reactants is determined in the following series of experiments. Reaction mixtures containing amounts of reagent specified in Table III below are prepared and reacted under the conditions specified. In each case the reaction mixture is vigorously agitated for the time indicated and at the temperature indicated. At the conclusion of the reaction period, a one ml. sample is withdrawn from each reaction mixture. Each sample is diluted immediately after its withdrawal with a dipotassium phosphate buffer solution (0.1 M; pH 6.2) to produce samples of 10-fold and 1-00-fold dilution for bioassay and the samples at each dilution are immediately cooled to minimize further reaction. The concentration of DL-oc-aminobenzylpenicillin in each case is determined by bioassay against B. sabtilis in the manner described in previous examples and the results are recorded in Table III.

The data of Table III indicates that the sodium citratecitric acid buffer is superior to the phosphate and succinate buffers and the best results With citrate buffer are obtained using 0.1 and 0.2 M buffer solutions. The ternperature in this series of experiments has little effect on the yields which also apparently level oif after a reaction period of one hour.

Experiment Nos. 13 and 30 demonstrate the bioactivity obtained from 6-aminopenicillanic acid alone since the ester is not introduced into the reaction mixture in these experiments. Experiment 14 shows that no bioactivity is detected in the reaction mixture which is subjected to ordinary reaction conditions in the absence of 6-aminopenicillanic acid. The term NR which appears in Table III indicates that no mg. was obtained in the bioassay procedure.

TABLE III 6-APA, Thio- Time, Temp., Cone, Expt. No. mg. phenol, Bufier, 2 ml. of listed butler hrs. 0. pH mcg. ABP/ml.

pH 10 10 6. 4 37 5. 45 1,150 10 10 6. 0 4 37 5. 7 1, 370 10 10 6. 0 4 37 5. 82 1,070 10 10 6. 0 4 37 6.05 750 10 1O 6. 0 4 37 4. 7 255 10 10 6. 0 4 37 5. 0 340 10 10 6. 0 4 37 5. 6 500 10 10 6. 0 4 37 5. 68 650 10 10 5. 4 37 5. 18 875 10 5. 5 4 37 5. 27 975 10 10 5. 5 4 37 5. 725 10 10 5. 5 4 37 5. 4 650 10 0 6. 0 4 37 6. 0 67. 5 0 10 6. 0 4 37 5. 88 NR 5 5 6. 0 0 28 5. 9 700 5 5 6. 0 O 37 5. 9 600 5 5 6. 0 0 5. 95 480 5 5 6. 0 28 800 5 5 6. 0 37 975 5 5 6. 0 45 825 5 5 6. 0 1 28 1, 150 5 5 6. 0 1 37 975 5 5 6. 0 1 45 1, 150 5 5 6. 0 2 28 1, 000 5 5 6. O 2 37 1, 200 5 5 6. 0 2 45 1, 200 5 5 6. 0 4 28 1, 150 5 5 6. 0 4 37 1, 400 5 5 6. 0 4 45 1, 550 5 0 6. 0 0 105 EXAMPLE 7 Eflect of Various Agents on Synthesis of a-Amino-Benzylpenicillin In each of the following twelve experiments, reaction mixtures containing the reagents specified in Table IV below are prepared. In experiments 1-8 the reaction mixtures are vigorously agitated at 37 C. for 4 hours and the pH of the reaction mixture at the beginning and end of the reaction period is recorded in Table IV. In experiments 9 12 the reaction mixtures were vigorously agitated at 37 C. for one hour with the reaction mixture being continuously extracted with diethyl ether. The concentration of DL-a-aminobenzylpenicillin in each reaction mixture at the conclusion of the reaction was determined by bioassay to be as recorded in Table IV.

In the following table DMAC means dimethylacetamide and EtOH means ethyl alcohol.

ether and vigorously agitated for the periods indicated in Table V below. At the times indicated below in Table V, a One ml. sample'of each reaction mixture is withdrawn, the ether removed and discarded, and solid sodium bicarbonate added to the sample. Each sample is diluted immediately after its withdrawal with a dipotassium phosphate buffer solution (0.1 M; pH 6.2) to produce samples of 10-fold and lOO-fold dilution for bioassay and the samples at each dilution are immediately cooled to minimize further reaction. The concentration of DL-a-aminm benzylpenicillin in each sample is then determined by bio- TABLE IV ThiopH Cone, Expt G-APA, phenol, Bufier system mcg.ABP/

No. mg. mg. ml.

0 hrs. 4 hrs.

10 10 2 ml. 0.1 M citrate, pH 6.0 5. 85 5.82 1, 200 10 10 21:11. 0.1 M citrate, pH 6.0 containing EtOH- a. 12 6. 3 600 10 10 2ml. 0.1 M citrate, pH 6.0 containing 20% DMAC 5. 35 5.6 725 10 10 2 ml. 0.1 M citrate, pH 6.0 5. 88 6.1 900 10 0 rln 5.6 5.88 67.5 10 0 2ml. 0.1 M citrate, pH 6.0 containing 50% EtOH 6. 3 6. 3 92. 5 10 0 21ml. 0.1 M citrate, pH 6.0 containing 20% DMAC 5. 38 5. 72. 5 10 0 2 1111.0.1 M citrate, pH 6.0 containing 20% urea 6. 0 6. 130 25 25 5.0 ml. 0.2 M acetic acid, pH 2.2 220 50 50 do 1,650 25 25 5.0 ml. 0.2 M acetic acid, pH 3.1 135 25 25 5.0 ml. 0.2 M acetic acid, pH 4.0 41o EXAMPLE 8 a assay against B. subtilis by the disc method and the results are as set forth in the Table V below. Preparation of DL-a-Aminobenzylpenicillin TABLE V Form reaction mixtures (prep. 1 Io. 1-4 below) each Reaction Cone. ABP containing 30 mg. 6-am1nopen1c1llamc acld, 30 mg. S-(DL- Preparation No. time, min. tgmgg/ml.) 2-phenylglycyl)thiophenol hydrochloride and 3 ml. of y M8533 0.2 M aqueous acetic acid, are prepared and adjusted to 1 15 1 340 H 2.2 with 10% hydrochloric acid. A fifth reaction 2' 30 1: 550 mixture (prep. No. 5 below) is prepared in the same i 28 928 manner except that the thioester is omitted. Each reac- 120 115 tion mixture is covered with from about 5 to 7 ml. diethyl The assay of preparation No. 5 shows bioactivity which is due to the unreacted 6-aminopenicillanic acid in the sample. The data shows that in this series of experiments, yields increased up to 60 minutes reaction time and thereafter decreased sharply. The yield at 60 minutes is 10 EXAMPLE 10 In the following experiments, each preparation contains 30 mg. of 6-am-inopenicillanic acid, S-(DL-2-pheny1- glycyl)thiophenol hydrochloride is indicated both in 5 equivalent to about 20% of the theoretical yield. i f amolmt amount Per and a Solvent, an as 1nd1cated in Table VII below. The solvents are as fol- EXAMPLE 9 lows: Solvent A is 0.2 M aqueous acetic acid adjusted In the following series of experiments, each preparato Pg Wlth 10% hydrochloric aold, Solvent D is tion contains 30 mg. 6-aminopenicillanic acid, S-(DL-2- 10 o oold, Solvents F and G qo solutlol'ls phenylglycyDthiophenol hydrochloride as indicated both oofltallllng f 2 5%, p y y, of acetic in total amount and amount per ml., and a solvent, all o P P VlgoIouSlY o o at as indicated in Table VI below. The solvents are as folmlmltes {11nd duflllg the Ioaotlon P io a one lows: Solvent A is 0.2 M aqueous acetic acid adjusted Sample 1$ W1thd1aW11 from each P P at 15 to pH 2.2 with 10% hydrochloric acid, solvent B is 90% 15 minutes, 30 {mums and at the end of the reaction period, acetic acid, solvent C is 75% acetic acid, and solvent D at 60 mlmltos- After o first Teaofiioll Poliod o 60 is 50% acetic acid. Each preparation is vigorously agimlmltos at P P are vigorously g t t d at 37 C f o hou d during th ti peat 37 C. for an additional reactlon period of 15 mlnutes riod, at 15 minutes, 30 minutes and 60 minutes, a one after whi on m sampl s are w1 hdrawn from each ml. sample is withdrawn from each preparation. Each p p pl sample is diluted immediately after its withdrawal with a e all. sample withdrawn as noted. above is dipotassium phosphate bufier solution (0.1 M; pH 6.2) to il to n n1- i l ly p Wi h r wal wi h a produce samples of 10-fold and 10() f 1d dil tio for saturated solut1on of sodium bicarbonate. A one ml. bioassay and the samples at each dilution are immediately portion of the ten-fold dilution with sodium bicarbonate cooled to minimize further reaction. The concentration solution is then diluted ten-fold with d-ipotassium phosof u-aminobenzylpenicillin in each sample is then deterphate buffer solution (0.1 M; pH 6.0). In the case of mined by bioassay against B. subtilis by the disc method at preparations 21-24, a one ml. portion of the IOO-fold 28 C. The experimental data is set forth in Table VI. dilution is again diluted with the butter solution to pro- TABLE VII Thiophenol Gone. in meg. ABP/ml. Flt-1?. Solvent Mg. M m1. 15 min min. 60min. 75 min.

TABLE VI duce a sample at LOGO-fold dilution and the concentration of a-am-inobenzylpen-icillin is determined from this sample. The sample of each preparation (one at 10-fold 'Ihhl 0.111 g.ABP1. Prcp.No. mp Ono Solvent one me Im dilution and one at IOO-fold dilution, at each of the M I 1. 15 30 l various time intervals) are then assayed against B. subg lm mm mm mm 60 zilis by the disc method and the concentration of a-ammo- 6 2 A 235 165 175 benzylpenic-ill-in is found to be as recorded in Table VII 35 11.7 A 280 235 325 above. From the experimental data, it can be seen that g 6- 2 2% 33,3 5 under the experimental conditions stated, the highest 6 2 A 97. 5 97. 5 87.5 yields of u-aminobenzylpenicillin are obtained using a 50% 3g i 238 332 aqueous solution of acetic acid as solvent.

0 o A 120 20 20 We claim: 2 5 g 38 1. A process for the preparation of u-alkylamino- 95 31.7 B 290 560 480 benzylpenicillin comprising contacting 6-aminopenicilg g E 298 lanic acid in an aqueous acidic reaction medium with a 35 11.7 C 8 22g 23g member selected from the group consisting of the com- 8 5 8 32 245 330 pounds having the formula 6 2 D 275 410 325 O 35 ii? a as .232 as: i

0 0 D 230 215 @ofi S@ 75 ENE HNR and

wherein R is a member selected from the group consist ing of hydrogen and lower alkyl and n is an integer from one to six inclusive and the acid addition salts of each of said compounds.

2. A process for the preparation of m-aminobenZyI- penicillin comprising contacting 6-aminopenicillanic acid in an aqueous acidic reaction media with a member selected from the group consisting of S-(u-aminophenyl- No references cited. 

1. A PROCESS FOR THE PREPRATION OF A-ALKYLAMINOBENZYLPENICILLIN COMPRISING CONTACTING 6-AMINOPENICILLANIC ACID IN AN AQUEOUS ACIDIC REACTION MEDIUM WITH A MEMBER SELECTED FROM THE GROUP CONSISTING OF THE COMPOUNDS HAVING THE FORMULA 